Functional genomic screen for modulators of ciliogenesis and cilium length

Abstract

Primary cilia are tiny hair-like structures expressed on the surface of eukaryotic cells. They participate in a wide range of biological processes such as sensing the extracellular environment to regulating numerous signalling pathways during development. Ciliary dysfunction has been linked to a group of human disorders known as ciliopathies. This study presents a functional genomic screen using RNA interference (RNAi) to identify human genes involved in control of ciliogenesis. Several positive and negative modulators of ciliogenesis with broad ranging functions were identified. Development of specific inhibitors that target key proteins may provide novel strategies to treat ciliopathies. Primary cilia are tiny hair-like structures expressed on the surface of eukaryotic cells. They participate in a range of processes, such as sensing the extracellular environment and regulating signalling pathways during development. Here, a functional genomic screen is presented that used RNA interference to identify human genes involved in controlling ciliogenesis. Several positive and negative ciliogenesis modulators with broad-ranging functions were found. Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways1,2. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities3. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance4. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell–cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.